The authors thank Dr. Amir Rattner for helpful comments on the manuscript. Supported by the Howard Hughes Medical Institute.

严格按照本指南中的美国国立卫生研究院实验动物的护理和使用的建议进行研究。所有的动物都按照批准的机构动物护理和使用委员会的约翰霍普金斯医疗机构（IACUC）协议MO11M29处理。咨询安乐死的方法核定的当地机构动物护理和使用委员会的指导方针。处理醛固色剂或有机溶剂时，应戴上手套，实验室外套，和护目镜。 材料1。准备<ol><li>浇SYLGARD板<ol><li>按照制造商的指示，用碱混合固化剂，并搅拌用塑料棒。 </li><li>移液器〜35毫升药液的Sylgard每10厘米直径的组织培养皿中。组织培养皿比标准菌菜更深;这提供了昆虫针垂直间隙。 </li><li>将二上在室温下水平表面畲族过夜。在混合过程中形成的气泡会消失。 </li><li>以下聚合中，存储的Sylgard板在室温下进行。 注意:的Sylgard板可在室温下保存多年，并重复使用多次，直至的Sylgard从板分开。 </li></ol></li><li>解决方案<ol><li>苯甲酸苄酯和苯甲醇（BBBA）。混合2卷苯甲酸苄酯和1体积的苯甲醇。存储在一个玻璃瓶中，用玻璃塞或特氟隆顶暗。 注意:使用BBBA应有机溶剂废物进行适当处理。不要让BBBA接触塑料。 </li><li>磷酸盐缓冲盐水（PBS）中。添加4克PFA至80毫升水中，加入0.1的1N氢氧化钠，搅拌，在加热板上在约70℃〜5分钟，直到煤灰完全溶解，然后加入10 ml 10X PBS中，使体积达到100毫升水。 注意:甲醛/ PFA和BBBA蒸气吸入应在有盖的容器保持这些解决方案最小化。甲醛/ PFA是一种致癌物质。 </li><li>油红O。准备一个0.5％的股票在异丙醇。在即将使用前用水稀释至0.3％的油红O的终浓度，然后通过0.2微米的过滤器进行过滤。 </li></ol></li></ol> 2，皮肤解剖，枷，及结算所<ol><li>背部皮肤的免疫染色和黑色素成像<ol><li>对于非常年幼的小鼠（ 如胎儿和出生后一天（P）0-P3的幼仔），异氟醚吸入安乐死的小鼠。异氟醚吸入或氯胺酮/赛拉嗪注射液颈椎脱位安乐死年纪较大的老鼠。 </li><li>用电动剃须刀和脱毛露脱毛的。对于小鼠年龄小于P8，这一步可以跳过。 </li><li>用剃刀刀片从沿每个侧翼的尾根部进行水平切割，通过对各肢的基部的背侧，则进行横向于耳朵和端ing在鼻子。 </li><li>轻轻剥离从下层组织程序从后方的皮肤保持戴手套的手指之间的皮肤，使其不被钳子夹住至前。当剥离皮肤的耳朵水平，先切断的耳朵用剪刀和进行特别慢可使皮肤撕裂在这个位置。 </li><li>从这点起，定向朝上内侧的皮肤上。脚皮肤的Sylgard与20〜昆虫针均匀地分布在周边;不要过度牵拉皮肤（ 图2B）。盖在10-20毫升的PBS皮肤。 </li><li>解剖相差使用成角度的或弯曲的钳朝向水平方向，以最小化该镊子将渗入皮肤的危险钳子的尖端与皮肤相关的脂肪和结缔组织。挤出已经被困在皮肤下轻轻滚动棉签在皮肤表面的气泡。 注:如果皮肤是怎么回事用于平面安装或免疫组织化学，除去尽可能多的结缔组织越好;如果皮肤将要使用基于黑色素形象化毛囊，少完全去除结缔组织是令人满意的。产前皮肤需要贴壁结缔组织极少"清洗"。 </li><li>通过加入20ml新鲜制备的4％PFA / PBS中，或10％福尔马林缓冲液中每10cm的Sylgard板固定钉扎皮肤（如皮肤，将用于黑色素成像或碱性磷酸酶（AP）组织化学）或20毫升新制备的2％PFA / PBS（如果皮肤将用于染色或可视化的转基因角蛋白（KRT）17-GFP 11），轻轻在冷室旋转过夜。第二天，在PBS洗为&gt; 10分钟。 注:标准福尔马林是37％的甲醛;因此，"10％的福尔马林"，是3.7％的甲醛。 </li><li>对于AP染色和免疫染色，继续在第3节，对于黑色素IMAGING，脱水，通过梯度乙醇系列皮肤（70％，95％，100％），一天的每个步骤中，在室温下温和水平旋转。去除残留的结缔组织。 </li><li>用在100％乙醇的皮肤，取出昆虫针，如果需要，用剪刀修剪的皮肤，除去最外围1-2毫米的皮肤与销孔。 </li><li>皮肤转移到一个直径10厘米的玻璃皿中，将两片玻璃显微镜载片上的皮肤，以防止翘曲，并添加20毫升BBBA的。 BBBA迅速变硬组织所以重要的是对皮肤的载玻片的重量下加入BBBA之前被平坦化。 </li><li>在接下来的几个小时，抬起滑离皮肤几秒钟，让BBBA洗在皮肤上。 注:与BBBA工作时戴上手套。取决于皮肤的年龄和位置可以有BBBA高达30％的收缩率。 </li></ol></li><li>足部皮肤的毛囊Patte的分析rning 注:年龄一般检查的范围为P1-P8。 <ol><li>安乐死后，切断英尺以上的踝关节，并通过脚底使沿腹面一个直切。 </li><li>轻轻地从下面的组织剥离皮肤，则进行在近侧至远侧方向。 </li><li>切位以释放在皮肤和针它SYLGARD与内表面朝上（ 图2C）。有在脚部皮肤最小的结缔组织。 </li><li>转基因KRT17-GFP的皮肤现在就可以成像。对于黑色素的成像进行固定，脱水和BBBA结算如2.1节所述。 </li></ol></li><li>足部皮肤的可视化皮脂腺<ol><li>安乐死后，通过摩擦毛发去除凝胶在皮肤表面用戴手套的手指和拇指除去毛发;等待10分钟。 注意:不要使用电动剃须刀，这将破坏脆弱的足部皮肤。 </li><li> W灰皮肤表面用自来水，解剖针和皮肤的Sylgard如2.2节所述。 </li><li>固定用1％PFA / PBS中30分钟，在室温下，然后在PBS洗。 注:有关脚，产后第一周之后，开发可视化的皮脂腺，进行分析与油红O的最佳年龄是P21，因为这标志着头发色素沉着的最低点在第一毛发周期12，因此，皮脂腺可以更容易地看出。用小白鼠这种分析可以在任何年龄进行。为了获得白化后代跨色素突变体小鼠酪氨酸酶的突变品系，如C57BL/6J-Tyr C-2J。 </li></ol></li><li>尾部皮肤准备的分析黑色素分布，毛囊方向，和皮脂腺可视化<ol><li>安乐死后，使周围的尾巴基部圆形切口，然后在运行沿其腹侧面尾部的长度的纵向狭缝。 </li> &lt;立&gt;揭去尾部皮肤牢牢抓住尾部骨和/或与一对钳子的，并与第二对钳子的皮肤结缔组织的前端开始在尖端。针尾皮肤的Sylgard。 </li><li>对于黑色素的分布和毛囊取向的分析，解决和处理如2.1节所述的皮肤。对皮脂腺的分析，切尾皮肤成一系列段〜0.5至1厘米的长度固定之前和孵化的皮肤片在PBS / 5mM EDTA的过夜，在37℃以削弱真皮-表皮粘附13。 </li><li>轻轻地从真皮层剥离表皮，脚表皮到的Sylgard（内面朝上），并固定在4％PFA / PBS中1小时，在室温下。用PBS冲洗，如下所述与油红O染色。 注意:一个电动剃须刀和/或脱毛凝胶可用于解剖较旧的尾部皮肤（ 如 P21）之前，但这种治疗方法是可选的，因为尾巴上的毛是不是作为致密作为对身体别处。 </li></ol></li></ol> 3，染色反应<ol><li>人胎盘碱性磷酸酶（AP）组织化学可视化基因标记的轴突乔木10,14,15（ 图3A，B） <ol><li>上的Sylgard PFA固定后，将皮肤固定在10厘米的玻璃盘，并在PBS / 1毫米MgCl 2淹没。轻轻旋转水浴中烹调，在70℃下进行90分钟至破坏内源磷酸酶活性。 </li><li>可视化的AP报告基因活性的组织化学与在室温下4-48小时培养中的0.1M Tris，pH值9.5，0.1M氯化钠，50毫摩尔MgCl 2，0.34μg/ ml的NBT和0.175微克/毫升BCIP，通常使用10-15毫升每P21背部皮肤染色溶液。监控解剖显微镜下的反应，注意不要让它继续下一步的最佳时间，作为判断的强度相对轴突染色的目视检查，以非特异性NBT沉积。 </li&gt; <li>停止洗涤用PBS/0.1％Tween-20中的在1小时的过程中3的变化，重新针的外观来的Sylgard，通过乙醇系列脱水的皮肤，然后将皮肤与转移到10cm的培养皿在反应BBBA。 注:BBBA慢慢溶解的NBT沉淀。在最初的几个小时中BBBA，沉淀NBT的罚款背景会溶解;作为一个结果，BBBA解决方案将变暗，并且皮肤会减轻。几个小时后变成新鲜BBBA。 </li><li>成像在AP染色的皮肤后，转移它在室温下用乙醇为&gt; 1小时，然后将其在-20℃储存在新鲜乙醇℃。 AP染色的外观可以被存储在这样的许多个月，无信号损失。 </li></ol></li><li>油红O染色，以可视化皮脂腺4（图3C-F） <ol><li>除去昆虫标签，并从板的Sylgard转移轻轻固定脚或尾部的皮肤到6孔组织培养板的孔中，与高达每孔2的皮肤样品。 5分钟，在室温下的水:用60:40异丙醇清洗。 </li><li>染色在2毫升0.3％油红O在60:40异丙醇:水（见1.2节）在室温下2小时。 </li><li>为5分钟，在室温下，接着用水洗涤水:用60:40的异丙醇洗涤。小心地修剪掉皮肤的边缘，其中的销孔，用细剪刀使皮肤的表面可制成尽可能平坦。 </li><li>放置在与外表面朝上滑动皮肤，添加几滴的水或含水介质的安装，盖上盖玻片，在皮肤上，然后轻轻地用胶带将盖玻片与滑动到进一步平坦化的皮肤。 </li></ol></li><li>平山4,16,17免疫染色（ 图3G，H） <ol><li>切PFA固定的皮肤（ 如 1厘米××0.5cm）上的一个矩形，标志着其方向与不对称剪裁（ 如夹右前角）。进行孵化和洗轻柔旋转在一个旋转的水平平台的步骤。在6井 - 或12 - 孔组织培养板，洗几次，用PBS以除去残留的PFA，洗净，用PBS超过5-8小时，在10〜变化用0.3％的Triton X-100（0.3％PBST）室温通透的皮肤。 </li><li>与第一抗体在0.3％PBST清洗，用5％山羊血清和20％DMSO的五天在室温下孵育。覆盖板与透明胶带或粘合剂的塑料，以消除蒸发损失的水井。 </li><li>用0.3％PBST中的10〜15改变洗涤过5-8小时，并用第二抗体孵育在0.3％PBST清洗，用5％山羊血清和20％DMSO的3天，在室温下进行。 </li><li>用0.3％PBST超过5-8小时10-15洗涤。 </li><li>脱水在25％，50％和75％甲醇的每个，然后3次在100％甲醇中的每个20分钟5分钟。 </li><li>明确在BBBA在室温下过夜。 注:平板安装肌肤年轻的染色比〜P1可如上所述进行，但以较短的温育和洗涤时间。例如，P1的皮肤可以用过夜培养初级抗体和几个小时的潜伏期在二级抗体进行免疫染色，并用介入的只有2-3小时洗0.3％PBST。如上面所指出的，更年轻的外观也足够薄，它们可以在不BBBA结算进行成像。 </li></ol></li><li>可视化默克尔细胞团荧光神经末梢染料吸收（ 图3I-L） AMI-43（绿色）和AM4-65（红色）是通过非选择性阳离子通道18进入细胞可固定的荧光染料。在生物皮肤，这些染料被选择性地吸收和浓缩默克尔细胞。 <ol><li>溶解1毫克2毫升的PBS中等分AM1-43或AM4-65，并储存于-80°C。 </li><li>皮下注入新生的幼崽与使用29号针头每克体重2-10微克染料。 </li><li>一天后，安乐死的小鼠和狄氏CT背侧皮肤。 </li><li>修正了在4％PFA / PBS的皮肤在4℃过夜，洗净，用PBS，并安装在3.2节中描述的皮肤。 </li></ol></li></ol> 4，成像和图像分析<ol><li>成像毛囊方向（ 图4） <ol><li>使用解剖显微镜图像背，脚，尾巴或皮肤，在玻璃皿淹没在BBBA，扁平载玻片下，仍然 - 这种方法最大限度地减少了显微镜BBBA污染。 </li><li>从下面照亮菜。为了尽量减少空间变化在整个视场中的光强度，提高了玻璃盘到一个高度〜10厘米解剖显微镜的标准工作表面上方，并放置一个漫射塑料或玻璃板在光源。 </li><li>返回到皮肤100​​％乙醇为长期贮存于-20℃。 注:将在BBBA皮肤更比一天造成的黑色素颗粒，打破了的一部分。一旦皮肤被BBBA对待它会变硬并从该点保持平稳前进。不同的外观可以在其前部和/或后部端部被编码的一系列凹口，从而使它们可以被存储在一起，以节省空间，在冰箱中。 </li></ol></li><li>成像和跟踪轴突乔木（ 图3A，B） <ol><li>将AP染色皮（节3.1.3）用于小蛋白凝胶类型的两块玻璃板之间。避免引入在板与皮肤之间的空气泡，然后小心地吸走多余的BBBA用纸巾。放置板皮板的上的显微镜载物台（ 图2D）的三明治。 </li><li>使用亮场或DIC照明用10X物镜和2微米或5微米的Z-步骤。使用机械化XY平台捕捉的XY图像的蒙太奇被缝合在一起创建一个单一的三维灰度数据集。 注:对于一个大的轴突乔木，该数据集可以是一个的大型为5 GB。 </li><li>进行手动，自动，或轴突乔木与任何各种软件包的半自动跟踪。 注:软件，如Neuromantic（http://www.reading.ac.uk/neuromantic/）是运行在PC环境中，是比较容易掌握10一个免费的神经元重建工具。 </li></ol></li></ol>

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M., Williams, J., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically-directed,+cell+type-specific+sparse+labeling+for+the+analysis+of+neuronal+morphology.">Genetically-directed, cell type-specific sparse labeling for the analysis of neuronal morphology.</a> PLoS One. 3, (2008).</li><li>Devenport, D., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+polarization+in+embryonic+epidermis+orchestrates+global+asymmetric+morphogenesis+of+hair+follicles.">Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles.</a> Nat. Cell Biol. 10, 1257-1268 (2008).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+organization+of+cutaneous+low-threshold+mechanosensory+neurons.">The functional organization of cutaneous low-threshold mechanosensory neurons.</a> Cell. 147, 1615-1627 (2011).</li><li>Meyers, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lighting+up+the+senses:+FM1-43+loading+of+sensory+cells+through+nonselective+ion+channels.">Lighting up the senses: FM1-43 loading of sensory cells through nonselective ion channels.</a> J. 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The authors have nothing to disclose.

上述的解剖方法的掌握，只需要耐心，一个稳定的手，和几个不错的清扫工具。背部皮肤剥离是相对容易的，但尾巴和脚的皮肤解剖 - 尤其是在出生后早期的年龄 - 是更具挑战性。在早期产前年龄（ 例如 ，E15之前），皮肤，很难不将其撕破删除。方便的是，对于生长和皮肤结构小鼠构图的很多研究中，感兴趣的事件发生时出生后，如在立毛肌19的生长的研究可见，例如。 <p class...

皮肤是身体中最大的器官，在躯体感觉，绝缘/体温调节和免疫防御1重要的功能。了解皮肤的发育和功能的细胞和分子基础已经因为皮肤作为一个生物系统及其与皮肤科相关的基本重要性的长期利益。哺乳动物的皮肤中含有多种多细胞结构，包括角质形成细胞分层层，真皮结缔组织，几种类型的毛囊，皮脂腺，立毛肌，血管，至少有十几个不同的类传入的（感觉）和传出神经的纤维（ 图1）。身体的不同区域与典型不同类型的皮肤有关。在大多数哺乳动物中，几乎整个身体表面覆盖有皮肤被密集地填充有毛囊。 [人类和无毛鼹鼠构成例外TH是的图案。头发是从手和脚，这也与专门的表皮图案（皮纹），外分泌腺和感觉神经末梢相关的掌面失踪。控制生长，分化和毛囊内的细胞空间排列的细胞和分子事件是特殊利益，因为每个毛囊展品，缩影，很多器官2的主要特征。这些功能包括干细胞的存在和干细胞小生境，正是编排细胞迁移，并从胚胎学上不同的部分多细胞结构的组装。 本文介绍了解剖方法，固定，标记和成像小鼠皮肤作为一个完整的二维表，被称为"整装"或"平山"的准备。自小鼠的皮肤较薄，也能够通过图像扁平的滑雪板的整个厚度N使用常规的共聚焦显微镜。平板安装的方法来成像哺乳动物皮肤在技术上是有利的，因为它绕过了需要物理切片，从而使结构将完全由光学切片重建。因为几乎整个皮肤被处理为一个单一的对象，平面安装方法也有利于身体表面的多个区域的成像，同时保持对位置和相对方位信息到主体轴。最后，对皮肤内的结构是典型地存在于被定期重复的是，从而便于从一个给定结构的多个代表图像的集合图案。这些特征是熟悉的在视网膜上，中枢神经系统，享有类似的优势，为神经元形态3的研究了二维部分谁工作神经生物学家。 这里所描述的平面安装的方法是特殊的utility表示学习表现出空间组织上的相对大的规模对皮肤的二维平面内的结构。大型空间组织的一个例子是毛囊和毛囊相关结构的协调极性- Merkel细胞簇，立毛肌，皮脂腺和神经末梢4。毛囊被定向在相对于皮肤的平面，并且位于皮肤的2维平面内的毛囊矢量的分量的角度通常表现出的取向相对于所述本体的轴是为每个精确地确定在身体上的位置。例如，从延髓背点毛囊尾鳍和头发上的脚的背侧面从近端指向远端。毛囊的方向是由平面细胞极性的信号控制（PCP，也称为组织极性5）。这个信号系统被发现在果蝇在一个小组核心PCP基因被发现，控制表皮的毛和硬毛的方向。的核心PCP基因三个哺乳动物直向同源物-型卷曲同源物6（FZD6，也被称为FZ6），钙粘蛋白EGF LAG 7通G-型受体1（CELSR1）和旺状2（VANGL2） -玩在哺乳动物类似的角色皮肤，协调毛囊的取向与主体轴。表明，在不存在PCP信令的主要缺陷是毛囊取向的初始随机化或解体，对毛囊的内在结构没有影响FZ6基因敲除小鼠（ - / - FZD6 tm1Nat，以下简称为FZ6）的研究6-8。第二个非PCP系统后的行为，促进毛囊附近，这导致生产大规模发模式，如旋涡及束的局部比对。 大规模的第二个例子皮肤内的空间组织被认为是在感觉轴突乔木的形态。感觉神经元支配的皮肤有其胞体在背根和三叉神经节。这些神经元的检测温度，疼痛，发痒，以及各种类型的机械变形的照射在皮肤和头发9。它们可以分为基于轴突直径和传导速度，终端神经末梢结构，受体，通道和其他分子的表达模式亚型。因为皮肤内神经支配的高密度，分析了包 ​​括可视化所有轴突（ 例如 ，抗神经丝免疫染色）或一个类的甚至全部的轴突（当单个细胞类型的特点是荧光记者表达看）通常揭示了一个致密的叠加轴突，使得它不可能确定单个轴的形​​态。为了解决这个问题，我们使用了极其稀疏基因定向升abeling生产背侧皮肤样本中，个人以及隔离轴突乔木是由一个组织化学记者，人胎盘碱性磷酸酶10表达的可视化。该方法允许个别轴突乔木形态的明确的视觉效果，并根据形态学标准躯体感觉神经元的类型的定义。

<table border="0" cellpadding="0" cellspacing="0" width="824">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Name of Material/ Equipment</td>
			<td style="width:256px;">Company</td>
			<td style="width:145px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-bromo-4-chloro-indolyl phosphate (BCIP)</td>
			<td>Roche</td>
			<td>11383221001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AM1-43</td>
			<td>Biotium</td>
			<td>70024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AM4-65</td>
			<td>Biotium</td>
			<td>70039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol</td>
			<td>Sigma</td>
			<td>402834</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate</td>
			<td>Sigma&#160;</td>
			<td>B-6630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Confocal microscope</td>
			<td style="width:256px;">Zeiss</td>
			<td style="width:145px;">LSM700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy3-alpha smooth muscle actin antibody</td>
			<td>Sigma&#160;</td>
			<td>C6198</td>
			<td>1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytokeratin-8&#160;</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-I-c</td>
			<td>1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection tools&#160;</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>scissors and forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electric razor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoromount G</td>
			<td>EM Sciences</td>
			<td>17984-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formalin</td>
			<td>Sigma</td>
			<td>HT501320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass dishes</td>
			<td>Pyrex&#160;</td>
			<td></td>
			<td>6 cm and 10 cm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass plates</td>
			<td>Amersham Biosciences</td>
			<td>SE202P-10</td>
			<td>10 cm x 8 cm x 1 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hair remover&#160;</td>
			<td>Nair</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horizontal rotating platform&#160;</td>
			<td style="width:256px;">Hoefer</td>
			<td style="width:145px;">PR250 Orbital shaker</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins</td>
			<td>Fine Science Tools&#160;</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine/xylazine</td>
			<td>Sigma</td>
			<td>K113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitroblue tetrazolium (NBT)</td>
			<td>Roche&#160;</td>
			<td>11383213001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oil Red O</td>
			<td>Sigma</td>
			<td>O0625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma&#160;</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Razor Blades</td>
			<td style="width:256px;">VWR</td>
			<td style="width:145px;">55411-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibodies&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td>Alexa-dye conjugated&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylgard-184</td>
			<td>Fisher Scientific</td>
			<td>NC9020938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plastic dishes</td>
			<td></td>
			<td></td>
			<td>10 cm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plates</td>
			<td></td>
			<td></td>
			<td>6- and 12-well&#160;</td>
		</tr>
	</tbody>
</table>

flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology

皮肤是一种高度非均质的组织。真皮内的结构包括毛囊，立毛肌，表皮特殊化（如Merkel细胞簇），皮脂腺，神经和神经末梢和毛细血管。这些结构的空间布置受到严格控制在微观尺度上 - 如看到的那样，例如，在在一个单一的毛囊细胞类型的有序排列 - 和在宏观尺度上 - 所看到的数以千计的头发在几乎相同的方向皮肤的局部区域​​内的毛囊。可视化这些结构而不物理切片的皮肤是因为这个器官的2维几何的可能。在这个协议中，我们证明了小鼠皮肤可解剖，固定，透，染色，明确作为一个完整的二维物体，一台安装。该协议允许对皮肤结构的可视化容易以其全部通过大面积的皮肤被选择的整个厚度iCal的切片和重建。这些结构中的图像也可以与左右位置和相对方位信息集成到主体轴。

皮肤铺片的明场成像可用于基于黑色素色素沉着（ 图4）的图像的皮肤感觉传入（ 图3A 10）和毛囊的图案。皮肤铺片的共焦成像可以被用来定义（1）Merkel细胞簇，可视化与抗细胞角蛋白6或带AM染料的吸收（ 图3I-L），（2）立毛肌，可视化与抗几何平滑肌肌动蛋白（ 图3G，3H），（3）皮脂腺，可视化与油红O（ 图3C-F）和（4）的...

Watch this Scientific Journal Video about Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology at JoVE.com

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

哺乳动物的皮肤中含有结构的多样化 - 如毛囊和神经末梢 - 表现出独特的空间组织模式。作为平坦安装分析皮肤借此组织的2维几何的优点，以产生皮肤结构的全厚的高清晰度图像。

小鼠皮肤及其毛囊图案和感觉轴突形态分析中的应用平山成像

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

flat-mount-imaging-mouse-skin-its-application-to-analysis-hair

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Neuroscience

21.3K 次观看.  Howard Hughes Medical Institute, Johns Hopkins University School of Medicine. 哺乳动物的皮肤中含有结构的多样化 - 如毛囊和神经末梢 - 表现出独特的空间组织模式。作为平坦安装分析皮肤借此组织的2维几何的优点，以产生皮肤结构的全厚的高清晰度图像。 

视频: Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

鼠标感觉神经支配的皮肤和四肢骨骼纤维组织的制备及免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

科研

神经科学

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

小鼠胚胎感觉轴突预测整个安装成像

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

机械刺激传入输出和神经末梢标签联合录制在小鼠毛囊披针形结局

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

在毛囊披针形结局生活神经末端标记的光学监测<em&gt;离体</em&gt;鼠耳皮肤

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

星爆无长突细胞的膜片钳在Deafferentated小鼠视网膜的平板安装准备

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.